1. Field of the Invention
The present invention relates to the cloning, expression and functional characterization of mammalian melanocortin receptor genes. The invention provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. The invention also provides a panel of such transformed mammalian cells expressing melanocortin receptors for screening compounds for receptor agonist and antagonist activity. The invention provides methods, for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify physiological function and behavior, particularly metabolism and feeding behavior.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, xcex1-melanocyte stimulating hormone (xcex1MSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones are also found in a variety of forms with unknown functions, for example, xcex3-melanocyte stimulating hormone (xcex3MSH), which has little or no ability to stimulate pigmentation (Ling et al., 1979, Life Sci. 25: 1773-1780; Slominski et al., 1992, Life Sci. 50: 1103-1108). A melanocortin receptor gene specific for each of the xcex1MSH, ACTH and xcex3MSH hormones has been discovered by some of the present inventors (see U.S. Pat. Nos. 5,280,112, 5,532,347 and U.S. application Ser. No. 08/044,812, incorporated by reference herein). In addition, two other melanocortin receptor genes have been discovered by some of the present inventors (see Lu et al, 1994, Nature 371: 799-802; Mountjoy et al, 1994, Molec. Endocrinol. 8: 1298-1308) and others (see Gantz et al., 1993, J. Biol. Chem. 268: 15174-15179 and Labbe et al., 1994, Biochem. 33: 4543-4549).
Along with the well-recognized activities of xcex1MSH in melanocytes and ACTH in adrenal and pituitary glands, the melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain and immune system, and bind to specific receptors in these tissues with a distinct pharmacology (see, Hanneman et al., in Peptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied and Jolles, 1982, Physiol. Rev. 62: 976-1059 for reviews). A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Shimuze, 1985, Yale J. Biol. Med. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.
Tatro and Reichlin, 1987, Endocrinology 121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.
Sola et al., 1989, J. Biol. Chem. 264: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.
Siegrist et al., 1991, J. Receptor Res. 11: 323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.
Cone and Mountjoy, U.S. patent application Ser. No. 07/866,979, filed Apr. 10, 1992, disclose the isolation of human and mouse xcex1-MSH receptor genes and uses thereof (incorporated herein by reference).
Cone and Mountjoy, U.S. patent application Ser. No. 07/866,560, filed Apr. 10, 1992, disclose the isolation of human and bovine ACTH receptor genes and uses thereof (incorporated herein by reference).
Mountjoy et al., 1992, Science 257: 1248-1251 disclose the isolation of cDNAs encoding mammalian ACTH and MSH receptor proteins.
POMC neurons are present in only two regions of the brain, the arcuate nucleus of the hypothalamus, and the nucleus of the solitary tract of the brain stem. Neurons from both sites project to a number of hypothalamic nuclei known to be important in feeding behavior, including the paraventricular nucleus, lateral hypothalamic area, and ventromedial hypothalamic nucleus. While previous reports have claimed both stimulatory and inhibitory effects of xcex1-MSH on feeding behavior (see Shimizu et al., 1989, Life Sci. 45: 543-552; Tsujii et al., 1989, Brian Res. Bull. 23: 165-169), knowledge of specific melanocortin receptors, their location within the central nervous system and the necessary pharmacological tools were not sufficiently developed at that time to allow the resolution of this issue. The present inventors have shown herein that a novel antagonist of the MC-3 and MC-4 melanocortin receptors can substantially increase food consumption in animals engaged in normal or fast-induced feeding behavior. This is consistent with expression of both MC-3 and MC-4 receptor mRNAs at these sites in in situ hybridization studies (Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci. USA 90: 8856-8860; Mountjoy et al., 1994, Molec. Endocrinol. 8: 1298-1308). Moreover, the regulation of arcuate nucleus POMC gene expression is consistent with an inhibitory role for POMC in feeding behavior. POMC mRNA levels are decreased following a fast (Bergendahl et al., 1992, Neuroendocrinol. 56: 913-920; Brady et al., 1990, Neuroendocrinol. 52: 441-447), and a significant diurnal variation in POMC mRNA levels in the arcuate nucleus is seen in rat, with the nadir occurring around the onset of nighttime feeding at 1800 hrs (Steiner et al., 1994, FASEB J. 8: 479-488).
Thus, the experimental evidence indicates that POMC neurons play an important role in tonic inhibition of feeding behavior, wherein obesity results from a chronic disruption of this inhibitory tone by antagonism of central melanocortin receptors in at least one animal model (agouti).
These results reveal for the first time a need in the art for a means for characterizing mammalian melanocortin receptor agonists and antagonists in vitro for the development of compounds that affect feeding behavior in animals.
The present invention provides a biological screening system for identifying and characterizing compounds that are agonists or antagonists of mammalian melanocortin receptors. The biological screening system of the invention comprises a panel of transformed mammalian cells comprising a recombinant expression construct encoding a mammalian melanocortin receptor, and expressing said receptor thereby. The invention provides such a panel of transformed mammalian cells wherein the panel comprises cells expressing each of type of mammalian melanocortin receptor. Thus, the invention also provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. Methods for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors, are also provided. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify metabolism and behavior in an animal, particularly feeding behavior.
Thus, the invention provides in a first aspect a biological screening panel for determining the melanocortin receptor agonist/antagonist profile of a test compound. The panel comprises a first mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the xcex1-MSH (MC-1) receptor. The panel also comprises a second mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the ACTH (MC-2) receptor. The panel also comprises a third mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-3 receptor. The panel also comprises a fourth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-4 receptor. The panel also comprises a fifth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-5 receptor. As provided by the invention, each mammalian cell expresses the melanocortin receptor encoded by the recombinant expression construct comprising said cell.
In preferred embodiments, the melanocortin receptors encoded by the recombinant expression constructs comprising the transformed mammalian cells comprising the panel are mouse MC-1 receptor (SEQ ID Nos.:3 and 4); human MC-I receptor (SEQ ID Nos.:5 and 6), human MC-2 (ACTH) receptor (SEQ ID Nos.:7 and 8), bovine MC-2 receptor (SEQ ID Nos.:9 and 10), rat MC-3 receptor (SEQ ID Nos.:11 and 12), human MC-4 receptor (SEQ ID Nos.:15 and 16) and human MC-5 receptor (SEQ ID Nos.:17 and 18).
In a second aspect, the invention provides a method for using the melanocortin receptor panel to identify and characterize test compounds as melanocortin receptor agonists and/or antagonists. In this embodiment, the method provided by the invention identifies a melanocortin receptor agonist, and comprises the steps of contacting each of the cells of the panel with a test compound to be characterized as an agonist of a mammalian melanocortin receptor and detecting binding of the test compound to each of the mammalian melanocortin receptors by assaying for a metabolite produced in the cells that bind the compound. In a preferred embodiment, the detected metabolite is cAMP.
In a preferred embodiment of this method, each of the cells of the panel of mammalian cells expressing mammalian melanocortin receptors further comprises a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site that is operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite. In preferred embodiments, said protein is xcex2-galactosidase, most preferably encoded by a nucleic acid comprising the recombinant expression construct identified as pCRE/xcex2-galactosidase (as disclosed in Chen et al., 1994, Analyt. Biochem. 226: 349-354). As provided by the invention, expression of the protein that produces the detectable metabolite is dependent on binding of the test compound to the melanocortin receptor expressed by each cell in the panel and the intracellular production of cAMP as a result. In this embodiment, cAMP production results in expression of a protein capable of producing a detectable metabolite, the protein most preferably being xcex2-galactosidase. In preferred embodiments, the detectable metabolite absorbs light to produce a colored product. Thus, this embodiment of the invention provides a panel of melanocortin receptor-expressing cells whereby melanocortin hormone binding results in the production of a colored product in proportion to the extent of cAMP production in the cell as a result of hormone receptor binding.
In another embodiment of this aspect of the invention is provided a method for characterizing a compound as an antagonist of a mammalian melanocortin receptor. In this embodiment, the method comprises the steps of contacting each of the cells of the panel with an agonist of the mammalian melanocortin receptor in an amount sufficient to produce a detectable amount of a metabolite produced in the cells that bind the agonist, in the presence or absence of a test compound to be characterized as an antagonist of a mammalian melanocortin receptor, and detecting the amount of the metabolite produced in each cell in the panel in the presence of the test compound with the amount of the metabolite produced in each cell in the panel in the absence. As provided by the assay, inhibition of the production of the detectable metabolite is used as an indication that the tested compound is a melanocortin receptor antagonist, which is further characterized quantitatively by the extent of said inhibition.
In a preferred embodiment of this method, each of the cells of the panel of mammalian cells expressing mammalian melanocortin receptors further comprises a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site that is operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite. In preferred embodiments, said protein is xcex2-galactosidase, most preferably encoded by a nucleic acid comprising the recombinant expression construct identified as pCRE/xcex2-galactosidase. As provided by the invention, expression of the protein that produces the detectable, metabolite is dependent on binding of the test compound to the melanocortin receptor expressed by each cell in the panel. In preferred embodiments, the detectable metabolite absorbs light to produce a colored product. Thus, this embodiment of the invention provides a panel of melanocortin receptor-expressing cells whereby melanocortin hormone binding results in the production of a colored product in proportion to the extent of cAMP production in the cell as a result of hormone receptor binding.
The invention also provides melanocortin receptor agonists identified by the methods and using the screening panel of the invention. In preferred embodiments, the agonist is an agonist of the MC-3 mammalian melanocortin receptor. In preferred embodiments, the agonist is an agonist of the MC-4 mammalian melanocortin receptor.
The invention provides melanocortin receptor antagonists identified by the methods and using the screening panel of the invention. In preferred embodiments, the antagonist is an antagonist of the MC-3 mammalian melanocortin receptor. In preferred embodiments, the antagonist is an antagonist of the MC-4 mammalian melanocortin receptor.
The invention also provides methods for characterizing mammalian melanocortin receptor agonists for the capacity to modify or influence metabolism and feeding behavior in an animal. In a first aspect, the invention provides a method for characterizing melanocortin receptor MC-3 or MC-4 agonists as inhibitors of feeding behavior in an animal, the method comprising the steps of providing food to an animal that has been deprived of food for at least 12 hours, with or without administering to the animal an MC-3 or MC-4 receptor agonist of the invention, and comparing the amount of food eaten by the animal after administration of the MC-3 or MC-4 receptor agonist with the amount of food eaten by the animal without administration of the MC-3 or MC-4 receptor agonist.
In another aspect, the invention provides a method for characterizing a melanocortin MC-3 or MC-4 receptor antagonist as a stimulator of feeding behavior in an animal. In this embodiment, the method comprises the steps of providing food to an animal not deprived of food for at least 12 hours, with or without administering to the animal an MC-3 or MC-4 receptor antagonist, immediately prior to the onset of darkness or nighttime, and comparing the amount of food eaten by the animal after administration of the MC-3 or MC-4 receptor antagonist with the amount of food eaten by the animal without administration of the MC-3 or MC-4 receptor antagonist.
Thus, the invention also provides methods for using certain of the melanocortin receptor agonists and antagonists for modifying feeding behavior in an animal. In a first aspect, the invention provides a method for stimulating feeding in an animal, the method comprising administering to the animal an MC-3 or MC-4 receptor antagonist. In a preferred embodiment, the antagonists are administered systemically. In additional embodiments, the antagonists are administered intracerebroventricularly.
In another aspect, the invention provides a method for inhibiting feeding in an animal, the method comprising administering to the animal an MC-3 or MC-4 receptor agonist. In a preferred embodiment, the agonists are administered systemically. In additional embodiments, the agonists are administered intracerebroventricularly. In yet another aspect, the invention provides mammalian melanocortin receptor agonists having the general formula:
A-B-C-D-E-F-G-amide
wherein A is an aliphatic amino acid residue, including for example Leu, Ile, Nle and Met, as well as analogues and substituted derivatives thereof; B is an acidic amino acid residue, including for example Asp and Glu; C is a basic amino acid residue, such as His; D is an aromatic amino acid residue having a D-conformation, including D-Phe, D-Tyr and substituted derivatives thereof; E is. a basic amino acid residue, for. example Arg, Lys, homoArg, homoLys, and analogues or substituted derivatives thereof; F is Trp or substituted derivatives thereof; and G is Lys, homoLys or a substituted derivative thereof. In the peptide embodiments of the melanocortin receptor agonists of the invention, the peptide is cyclized by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. In preferred embodiments, the melanocortin receptor agonists of the invention are agonists of the MC-3 or MC-4 receptor.
The invention also provides mammalian melanocortin receptor antagonists having the general formula:
A-B-C-D-E-F-G-amide
wherein A is an aliphatic amino acid residue, including for example Leu, Ile, Nle and Met, as well as analogues and substituted derivatives thereof; B is an acidic amino acid residue, including for example Asp and Glu; C is a basic amino acid residue, such as His; D is an aromatic amino acid residue having D-conformation, including D-Nal and substituted derivatives thereof; E is a basic amino acid residue, for example Arg, Lys, homoArg, homoLys, and analogues or substituted derivatives thereof; F is Trp or substituted derivatives thereof; and G is Lys, homoLys or a substituted derivative thereof. In the peptide embodiments of the melanocortin receptor antagonists of the invention, the peptide is cyclized by the formation of an amide bond between the side chain carboxyl group of the Asp or Glu residue at position B in the peptide, and the side chain amino group of the Lys or homoLys residue at position G. In preferred embodiments, the melanocortin receptor antagonists of the invention are agonists of the MC-3 or MC-4 receptor.
It is an advantage of the present invention that it provides an in vitro screening method for characterizing compounds having melanocortin receptor activities that relate to feeding behavior in animals. Specifically, the invention advantageously provides means and methods for identifying compounds having melanocortin receptor agonist and/or antagonist activity that have been associated with either stimulating or inhibiting feeding behavior when administered to an animal. The invention thus provides an economical first step in screening compounds for the capacity to affect feeding behavior, including synthetic, peptidomimetic or organomimetic derivatives of melanocortin receptor agonists or antagonists as disclosed herein or elsewhere.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.